Contrast-enhanced computed tomography offers a nondestructive approach to studying adipose tissue in 3D. Several contrast-enhancing staining agents (CESAs) have been explored, whereof osmium tetroxide (OsO4) is the most popular nowadays. However, due to the toxicity and volatility of the conventional OsO4, alternative CESAs with similar staining properties were desired. Hf-WD 1:2 POM and Hexabrix have proven effective for structural analysis of adipocytes using contrast-enhanced computed tomography but fail to provide chemical information. This study introduces isotonic Lugol's iodine (IL) as an alternative CESA for adipose tissue analysis, comparing its staining potential with Hf-WD 1:2 POM and Hexabrix in murine caudal vertebrae and bovine muscle tissue strips. Single and sequential staining protocols were compared to assess the maximization of information extraction from each sample. The study investigated interactions, distribution, and reactivity of iodine species towards biomolecules using simplified model systems and assesses the potential of the CESA to provide chemical information. (Bio)chemical analyses on whole tissues revealed that differences in adipocyte gray values post-IL staining were associated with chemical distinctions between bovine muscle tissue and murine caudal vertebrae. More specific, a difference in the degree of unsaturation of fatty acids was identified as a likely contributor, though not the sole determinant of gray value differences. This research sheds light on the potential of IL as a CESA, offering both structural and chemical insights into adipose tissue composition.

Institute of Mechanics Materials and Civil Engineering Mechatronic Electrical Energy and Dynamic Systems UCLouvain Louvain la Neuve Belgium; Pole of Morphology Institute of Experimental and Clinical Research UCLouvain Brussels Belgium; Department Materials Engineering KU Leuven Leuven Belgium

Laboratory of Molecular Physiology of Bone Institute of Physiology of the Czech Academy of Sciences Prague Czech Republic

Laboratory of Molecular Physiology of Bone Institute of Physiology of the Czech Academy of Sciences Prague Czech Republic; Faculty of Science Charles University Prague Czech Republic

Laboratory of Translational Metabolism Institute of Physiology of the Czech Academy of Sciences Prague Czech Republic

Membrane Separations Adsorption Catalysis and Spectroscopy for Sustainable Solutions Department of Microbial and Molecular Systems KU Leuven Leuven Belgium

MolDesignS Sustainable Chemistry for Metals and Molecules Department of Chemistry KU Leuven Leuven Belgium

MolDesignS Sustainable Chemistry for Metals and Molecules Department of Chemistry KU Leuven Leuven Belgium; Institute of Mechanics Materials and Civil Engineering Mechatronic Electrical Energy and Dynamic Systems UCLouvain Louvain la Neuve Belgium; Pole of Morphology Institute of Experimental and Clinical Research UCLouvain Brussels Belgium

Molecular Imaging and Photonics Department of Chemistry KU Leuven Leuven Belgium

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